The present invention relates to a bactericide (and a fungicide) originated from natural products. More specifically, the present invention relates to a bactericide (and a fungicide) containing an extract obtained by extraction from leaves of eucalyptus plants into a polar organic solvent and chitosan, or a bactericide (and a fungicide) containing glycerol fatty acid ester in addition to the above-mentioned components, which has strong and persistent bactericidal (or fungicidal) power and high safety.
The bactericide (and a fungicide) of the present invention can be used at home, restaurants, medical facilities, old-age homes, abattoir facilities, cattle sheds, chicken houses, and the like. Particularly, the present invention is useful as a bactericide (and a fungicide) for preventing an acne, athlete""s foot.
There are about six hundred species of eucalyptus plants, of which an essential oil or an ethanol extract or the like are used as pharmaceuticals, quasi-drugs, food additives and perfumery, such as pharmaceuticals for asthma, preservatives, aromatic substances (Hiroyuki Nishimura, xe2x80x9cMirai no Seibutsu Shigen Yukarixe2x80x9d, 1987). It has been reported that cineole that is a major component of eucalyptus essential oil has an effect as antiseptics, or enhances an effect of antibacterial substance (bisbiguanide compound) (Japanese Patent Application Laid-open publication (KOKAI) publication No. Sho 62-289511). It has been reported that grandinol isolated from an organic solvent extract of Eucalyptus perriniana (Agric. Biol. Chem., 54, 1, 231, 1990) and Macrocarpal-A isolated from Eucalyptus macrocarpa (Agric. Biol. Chem., 54, 12, 3221) have antibacterial activity against Staphylococcus aureus and Bacillus subtilis. 
Chitosan is a compound produced by deacetylating chitin contained in a carapace of a crab or the like, and is also used as a food additive. It has been reported that chitosan has an antibacterial activity against Fusarium, Escherichia coli, Staphylococcus aureus, Bacillus subtilis or the like (Experimental Mycology 8, 276, 1984, Food chemical 2, 22, 1988).
There have been reported examples of a synergy effect achieved by mixing two or more of antibacterial components, such as rifampicin and chitosan (Japanese Patent Application Laid-open publication (KOKAI) No.59-46208), sulfonamides and chitosan (Japanese Patent Application Laid-open publication (KOKAI) No.59-46223), chitosan and ascorbic acid (Japanese Patent Application Laid-open publication (KOKAI) No. 2-35065), organic solvent extract of eucalyptus (species is not known) and Sodium alkyl sulfate (Japanese Patent Application Laid-open publication (KOKAI) No.58-39615). However, there has not been reported a combination of eucalyptus and chitosan. There have been reported a combination of an extract of eucalyptus and chitosan for a preservative for a flower (Japanese Patent Application Laid-open publication (KOKAI) No.4-316506), pesticide for plants (Japanese Patent Application Laid-open publication (KOKAI) No.7-033602). However, there has not been reported a synergy effect of antibacterial activity achieved by such a combination.
Acne is caused by propagation of Propionibacterium acnes present in sebaceous crypt. Athlete""s foot is caused by infection with Trichophyton mentagrophytes. Accordingly, if Propionibacterium acnes and Trichophyton mentagrophytes can be sterilized efficiently, an acne and athlete""s foot can be prevented.
Recently, hospital acquired infection caused by pathogenic bacterium such as MRSA (methicillin-resistant Staphylococcus aureus), VRE (vancomycin-resistant Enterococcus) or the like has been increased at medical facilities and old-age homes. Accordingly, it is an urgent subject to sterilize this pathogenic bacterium efficiently.
In an abattoir, there is a problem of contamination of apparatuses (a mincing machine, a silent cutter, a slicer, mixer, filler, a meat dehydration machine or the like) and facilities for processing meat with food poisoning bacterium such as Escherichia coli, Salmonella enteritidis, Staphylococcus aureus or the like.
In a conventional abattoir, sodium hypochlorite is used as a bactericide during a sterilizing process of meat. Namely, meat such as carcass (dressed carcass) is immersed in a solution of sodium hypochlorite for a certain time. However, according to the method, there is caused a chemical reaction of sodium hypochlorite and proteins in meat or gravy seeping from meat, resulting in lowering or vanishment of a bactericidal effect. Furthermore, there is a problem of safety for a human body of reaction products of sodium hypochlorite adhering to meat.
For the above-mentioned reasons, there has been requested a bactericide that has a bactericidal power that is not lowered during sterilization of meat, high safety for a human body, and a long lasting bactericidal power. Such a bactericide will keep meat fresh for a long time, and make it possible to decrease or prevent degradation of products in transit.
Furthermore, prevention of contamination in an abattoir can also be achieved by reducing contamination of livestock itself with pathogenic bacteria or fungus. One of the methods comprises sterilization of a cattle shed. Escherichia coli that is especially problematic, lives in livestock such as cattle, sheep, pig or the like. Especially, a cattle is contaminated at higher rate. Furthermore, Salmonella enteritidis is also problematic, spreading through chickens or eggs of hens. For example, if a cattle shed is contaminated with pathogenic microbe, contamination may be enlarged through livestock or hands of people who touch the livestock or the like in a cattle shed. Accordingly, it is also important to sterilize a cattle shed in order to prevent pathogenic microbe efficiently.
In stockyard for cattle, pig, chicken or the like, there occur problems such as contamination of fodder with bacteria, propagation of the bacteria in fodder or the like. If fodder is contaminated with bacteria, not only it is harmful for animals, but also contamination may be enlarged through excrementum of the animal as a source of contamination. Conventionally, fodder to which antibiotics were added were invented in order to hasten growth of animals. However, there are problems of appearance of resistant bacterium or residual antibiotics, and thus use of antibiotics is now restricted. Antibiotics are used also for fish to prevent or treat infection of pathogenic bacterium, and it is also problematic.
Recently, incidence of food poison, especially those caused by salmonella, has been increased year by year, in eggs of hen or processed foods of eggs. It is possible to decrease or prevent the contamination by sterilizing shell of eggs.
It is reported that urea-splitting bacterium is relevant to diaper rash. Accordingly, it is assumed that diaper rash can be decreased or prevented by mixing bactericide (or a fungicide) into a diaper.
As bactericides against various bacteria mentioned above, there are used alcohol, synthetic bactericides, chloric agent such as sodium hypochlorite or the like. Alcohol has a problem of insufficient persistency of bactericidal power. Synthetic bactericides and chloric agents have problem of a lack of safety for a human body. Highly safe bactericides against the above-mentioned pathogenic microbe are requested, since they may be in contact with skin of human or animals, or orally ingested.
Accordingly, an object of the present invention is to provide a bactericide (and a fungicide) that is highly safe and has strong bactericidal power against the above-mentioned pathogenic microbe even when used in low concentration, which is not reduced during a sterilizing process of meat, and that can be used repeatedly in a sterilizing process.
The inventors of the present invention have found that a bactericide (and a fungicide) containing a polar organic solvent extract of eucalyptus and chitosan has a strong bactericidal (or fungicidal) power in low concentration due to a synergy effect of extract of eucalyptus and chitosan, and also it has bactericidal (or fungicidal) power that can last for a long time, and is not lowered when used as a bactericide for meat (including meat products such as ham, sausage or the like). Furthermore, it has been found that, when glycerol fatty acid ester is added to the bactericide (and a fungicide) having the above-mentioned components, persistency of the bactericidal (or fungicidal) power is improved, and antifungal power of glycerol fatty acid ester is enhanced.
Namely, the present invention relates to a bactericide (and a fungicide) basically comprising a polar solvent extract of leaves of eucalyptus plants and chitosan, and if desired, additionally containing glycerol fatty acid ester, wherein the polar solvent is chosen from the group consisting of lower alcohols and glycols. Especially, the polar solvent extract of leaves of eucalyptus plant is a fraction extracted by extraction into a polar organic solvent from leaves of eucalyptus plants wherein essential oil is previously removed by degreasing with a non-polar organic solvent or steam distillation. Examples of the polar organic solvent used herein include: solvent chosen from the group consisting of halogenated hydrocarbon, ethers, fatty acid esters, ketones and lower alcohols or a mixture thereof. Example of the above-mentioned non-polar organic solvent can be alkanes.
An example of an extract from leaves of eucalyptus plant into a polar solvent can be the extract containing as a major bactericidal (or fungicidal) component a dihydrochalcone compound represented by the following formula (I): 
The bactericide (and the fungicide) of the present invention can be used for sterilizing medical facilities, abattoir facilities, cattle sheds, chicken houses, egg shells, meat or the like. It can also be used for affording bactericidal power to fodder, wet tissue paper, diaper or the like by mixing it therein.
The bactericide (and the fungicide) of the present invention is highly safe for human body, since it contains as active ingredients natural products that are known to be safe.
The word xe2x80x9csterilizingxe2x80x9d in the present invention means both of killing microbes such as bacteria and inhibiting propagation thereof. The word xe2x80x9cabattoir facilitiesxe2x80x9d means equipment or apparatuses installed in an abattoir, as well as cooking devices such as cutlery or the like, table or the like used for processing meat.
The present invention will be explained below in more detail.
(1) Polar Organic Solvent Extract of Eucalyptus Plants
A polar organic solvent extract of eucalyptus used for the bactericide (and the fungicide) of the present invention (hereunder referred to as xe2x80x9ceucalyptus extractxe2x80x9d) is an extract obtained by extraction from leaves of eucalyptus plants into a polar organic solvent.
Leaves to be used as a raw material can be any leaves of plants belonged to eucalyptus, for example, Eucalyptus grandis, Eucalyptus botryoides, Eucalyptus globulus, Eucalyptus camaldulensis, Eucalyptus crebra, Eucalyptus maculata, Eucalyptus viminalis or the like. These leaves of eucalyptus can be those obtained from only one specie of the plants, or those obtained from two or more species of the plants.
Extraction from such leaves of eucalyptus plants is conducted with a polar organic solvent. The leaves are subjected to preliminary treatment, for example crashing of leaves to a suitable size, powdering of them, or the like, so that solvent extraction can be easily conducted. Examples of the polar organic solvent include: halogenated hydrocarbons such as chloroform, dichloro methane, dichloro ethane, trichloro ethane, or the like; ethers such as methyl ether, ethyl ether, tetrahydrofuran, dioxane or the like; esters of lower fatty acids such as methyl acetate, ethyl acetate, butyl acetate or the like; ketones such as acetone, methyl ethyl ketone or the like; lower alcohols such as methanol, ethanol, propanol or the like; glycols such as propylene glycol, butylene glycol or the like. These solvents can be alone or as a mixed solvent consisting of two, three or more kinds of any solvents. Among of the above solvents, ethyl acetate, acetone, ethanol, propylene glycol, butylene glycol are preferable, and ethanol or propylene glycol are more preferable, from the standpoint of the bactericidal activity of the resultant extract.
The method for extraction can be any methods generally conducted, including a method consisting of immersing leaves of eucalyptus as a raw material in a polar organic solvent for a long time, a method consisting of heating the leaves in a polar organic solvent with stirring at a temperature not higher than a boiling point of the polar organic solvent, and filtrating it to obtain the extract. The extract thus obtained is preferably concentrated by vacuum concentration or the like.
In order to obtain an eucalyptus extract having high bactericidal activity, the above-mentioned operation of extraction and separation are preferably conducted, not by direct extraction from leaves of eucalyptus into a polar organic solvent, but by drying the leaves of eucalyptus, degreasing them with a non-polar organic solvent to remove essential oil, and extracting the component from them into a polar organic solvent. Essential oil is removed in a process of degreasing with non-polar organic solvent. Such an operation can also be conducted by adding a polar solvent for extraction to a residue after removing essential oil by steam distillation. The above mentioned non-polar solvent can be alkanes such as hexane, pentane, heptane or the like.
However, the degreasing operation is not essential for the present invention. A fraction obtained by directly extraction from leaves of eucalyptus plant into a polar organic solvent can also be used as a bactericide (or a fungicide) of the present invention. In that case, a polar organic solvent can be lower alcohols, or glycols such as propylene glycol, butylene glycol or the like.
Comprehensively appreciating the bactericidal activity of extract of eucalyptus leaves and yield thereof, a method that can provide an extract having a strong bactericidal activity and enable high yield of the extract is a method wherein hexane is used as a non-polar solvent for degleasing, and ethanol is used as a solvent for extraction.
An extract of leaves of Eucalyptus maculata contains as a bactericidal compound a novel dihydrochalcone compound represented by the formula (I) shown above and known flavone compounds (eucalyptin and 8-desmethyl-eucalyptin) (Aust. J. Chem. 17, 692, 1964; Aust. J. Chem. 17, 464, 1964)). It is assumed that these compounds or analogues thereof may be used as an index in preparation of an extract of eucalyptus used for the present invention, although other compounds having a bactericidal activity may be contained in leaves of eucalyptus. As an embodiment of the extract of eucalyptus used for the present invention, there can be exemplified an extract containing at least one or more kinds of the above-mentioned dihydrochalcone compound, flavone compounds or analogues thereof as main bactericidal components, and purified compounds and analogues thereof.
(2) Chitosan
Chitosan (polyxcex2-1,4-glucosamin) is deacetylated chitin, which can be obtained, for example, by deacetylating chitin contained in a carapace of crustacean such as a crab, a lobster or the like, exoskeleton of insects in high concentration hot alkali solution. Alternatively, chitosan can also be obtained by culturing chitosan producing bacterium. Chitosan is also commercially available.
Molecular weight of chitosan used in the present invention is not limited, but preferably it has low viscosity, as about 5 to 50 cp (0.5% chitosan concentration). Chitosan derivatives of which aqueous solubility is increased, such as chitosan oligosaccharide, chitosan lactate, chitosan chloride or the like can also be used. However, in order to prepare a bactericide (or a fungicide) having a strong bactericidal (or a fungicidal) activity, chitosan is preferably used.
(3) Composition
The content of the above-mentioned eucalyptus extract and chitosan can be varied suitably depending on a way of use or a dosage form thereof. For example, the content of them in total is 0.0001 to 10% by weight, preferably 0.001 to 1.0% by weight. When non-volatile glycols such as propylene glycol, butylene glycol or the like are used as a solvent for extraction from leaves of eucalyptus, an eucalyptus extract is contained, for example, at a concentration of 0.1 to 10% by volume. At a higher concentration, aroma of a bactericide (or a fungicide) is adversely affected. At a lower concentration, sufficient effect is hardly achieved. Accordingly, the above-mentioned range is preferable. Eucalyptus extract and chitosan can be mixed at any ratio as far as a concentration thereof is in the above-mentioned range. Acids for dissolving chitosan (lactic acid, acetic acid or the like) can be added at any concentration so that chitosan can be dissolved.
(4) Bactericide (and a fungicide) to which Glycerol Fatty Acid Ester is Added
According to the present invention, glycerol fatty acid ester can be added to the above-mentioned bactericide (and a fungicide) having a composition comprising eucalyptus extract and chitosan. In that case, 0.0001 to 20% by weight, preferably 0.001 to 1.0% by weight of glycerol fatty acid ester can be added to the above-mentioned composition comprising eucalyptus extract and chitosan.
When the above-mentioned bactericide is used as a bactericide for meat, components seeping from meat during sterilization increase pH of the bactericide. If pH is more than 5.5, a bactericidal power is lowered. Accordingly, acids such as lactic acid, acetic acid or the like or a pH control agent are added to maintain pH to be 5.5 or less, preferably 5.0 or less. The acids or the pH control agents can be added in advance to the bactericide at high concentration, or the acids can be continuously added during sterilization to maintain pH to be 5.5 or less.
There can be mixed in the bactericide (and a fungicide) of the present invention other components that are generally used for foods, pharmaceuticals, quasi-drugs, cosmetics or the like, acidity control agents, stabilizers, surfactants, antioxidants or the like, as far as the effect of the present invention is not deteriorated, and two or more of the components can be mixed therein. Furthermore, the bactericide (and a fungicide) of the present invention can be used together with other bactericides (or fungicides) to enhance bactericidal (or fungicidal) effect.
Examples of the components generally used for foods, pharmaceuticals, quasi-drugs, cosmetics or the like include: surfactants (anionic, cationic, ampholytic surfactants or non-ionic surfactants), antioxidant (stearate, nordihydro guasereten acid, dibutyl hydroxyl toluene, butyl hydroxy anisole, p-hydroxyl anisole, propyl gallate, sesamol, sesamolin, gossypol or the like), humectant (propylene glycol, 1,3-butylene glycol, polyethylene glycol, glycerin, chondroitin sulfuric acid and salt thereof, hyaluronic acid and salt thereof, sodium lactate, royal jelly extract or the like). Furthermore, collagen, lower alcohols, polyhydric alcohols, water soluble polymers, pH control agents, flavoring agents, restorative agents, stabilizers, extracts of animals or plants, proteins of animals or plants and decomposed products thereof, polysaccharides of animals or plants and a decomposed products thereof, glycoproteins of animals or plants and decomposed products thereof, microorganism culture metabolites, amino acids and salts thereof, deodorizing agents, emulsifying agents can also be mixed therein and used.
Examples of the acidity controls and the stabilizers for the functions include: adipic acid, citric acid, sodium citrate, glycine, fatty acid glyceride, glucono-xcex4-lactone, gluconic acid, succinic acid, sodium hydrogen succinate, sodium succinate, acetic acid, sodium acetate, DL-tartaric acid, L-tartaric acid, DL-sodium tartrate, L-sodium tartrate, carbonates, carbon dioxide, lactic acid, sodium lactate, fumaric acid, monosodium fumarate, lysozyme, DL-malic acid, DL-sodium malate, phosphoric acid, phosphates, polymerized phosphates, itaconic acid, phytic acid and the like.
Examples of the surfactants include: fatty acid esters such as glycerol monostearate, trioleic acid polyglycerol or the like, organic acid monoglyceride, propylene glycol fatty acid ester, sorbitan fatty acid ester, sugar fatty acid ester, lecithin, lysolecithin, polyethylene glycol, polyoxy alkyl ether, polyoxy ethylene polyamine, alkyl polyoxy ethylene sulfate, a salt of alkyl sulfate ester, a salt of acyl methyl taurine, N-acyl glutamate, alkyl amide betain and the like.
Examples of the antioxidants include catechin, tocopherol, propolis, ellagic acid, extracts of animals or plants (sage, The Japanese parsley, rosemary or the like).
Examples of the bactericides include any of substances or extracts having bactericidal activity, such as hinokitiol, triclosan, cetylpyridinium chloride, chlorhexidine gluconate, extracts of animals or plants, essential oils or the like.
As described above, due to the synergistic effect of the eucalyptus extract and chitosan, the bactericide containing eucalyptus plant extract and chitosan has a strong bactericidal power against methicillin-resistant Staphylococcus aureus(MRSA), Staphylococcu aureus, Escherichia coli, Salmonella typhimurium, Salmonella enteritidis, Enterococcus faecalis, Pseudomonas putida, Bacillus subtilis, Bacillus cereus, Vibrio parahaemolyticus, Arthrobacter globiformis, Brevibacterium linens, Proteus vulgaris, Propionobacterium acnes, Trichophyton mentagrphytes, and the bactericidal power is persistent. Both of the eucalyptus extract and chitosan have high safety. Accordingly, if ingredients having high safety are chosen as the other ingredients, a bactericide having high safety can be provided.
The fungicide wherein glycerol fatty acid ester is added to the composition containing the above-mentioned eucalyptus extract and chitosan may have an effect against fungi such as Aspergillus niger, Penicillium citrinum or the like, due to the synergistic effect of the eucalyptus extract and glycerol fatty acid ester. If the fungicide of the present invention is prepared as a mixture in ethanol and water, an effect to the above fungi can also be achieved, since an instantaneous bactericidal power of ethanol is afforded thereto.
(5) Utility
The preparation form of the bactericide (and fungicide) of the present invention is not limited. It can be selected depending on an application form such as a liquid agent, a spray agent, a paste agent, a mousse agent or the like. It can also be defined appropriately depending on an application method such as spraying, dipping, coating, wiping-up or the like. The bactericide (or a fungicide) may be a spray agent. It can be used at a kitchen of restaurants or general home or the like. When it is in the form of liquid, an eucalyptus extract and chitosan are dissolved in suitable solvent to provide a bactericide (or a fungicide) solution. Examples of the solvent include: mixture of ethanol and water (at any mixing ratio) to which an acid such as lactic acid, acetic acid is added in order to dissolve chitosan. However, when the bactericide is used as a bactericide for meats, concentration of alcohol is preferably in the range of about 0 to 10%, since an alcohol at high concentration may denature proteins. The above-mentioned acid is not necessary to be used, when chitosan oligosaccharides having an increased water solubility or chitosan derivatives having an increased water solubility are used.
In the case that the bactericide of the present invention is used in a step of sterilizing meat, it can be used repeatedly in the sterilizing step, since a bactericidal power thereof is not lowered. If the bactericide is used for sterilizing treatment of meat, it can keep meat fresh for a long time, since it can inhibit propagation of the bacterium on the surface of the meat for a long time.
When the bactericide is used as a bactericide of egg shells of birds, the eggs on which the bactericide of the present invention is adhered can be distributed.
When the bactericide (or the fungicide) is used as spray agents, the storage containers can be those enabling sparay of the bactericide. For example, it is the sprayer container due to aerosol method, trigger method or the like. When the container of aerosol method is used, an agent for spraying like carbonic acid gas, nitrogen gas or dimethyl ether is added.